Microbiology 101 for Small Meat Processors

Small Meat Processors September 13, 2012 Print Friendly and PDF

A Niche Meat Processor Assistance Network webinar

Overview

N-60 sampling. Multi drug resistance. Pathogen v. adulterant. Non-0157 STECs. MDR Salmonella strains. Gram negative v. gram positive organisms. CFU, APC, PCR, PFGE.  If you’re a meat processor, you pretty much have to know all this stuff. And if you work with a meat processor, you should probably understand the basics so you know what your processor is up against. On this webinar, two Pennsylvania State University food scientists, who have many years experience working with small meat processors, explain the basic microbiological terms that processors – and their customers – need to understand.

The material was presented in four sections:

  • Microorganisms in Food
  • Pathogens
  • Spoilage Organisms
  • Microbial Analysis
 
Date: Wednesday, February 1, 2012
Time: 1pm Eastern/10am Pacific
Duration: 100 minutes
 
Speakers: 
  • Catherine Cutter, Ph.D., Associate Professor and Food Safety Extension Specialist, Pennsylvania State University; Dr. Cutter's website
  • Martin Bucknavage, Senior Food Safety Extension Associate, Pennsylvania State University; Mr. Bucknavage's website

 

Presentation Slides

 

Recording of the Webinar

 

Transcript of the Webinar

Introduction, Lauren Gwin, NMPAN

Welcome to the Microbiology 101 for Small Meat Processors, organized by the Niche Meat Processor Assistance Network.

Cathy Cutter is an Associate Professor and Food Safety Extension Specialist. She focuses on muscle foods and she is Chair of the Food Safety Impact Group at Pennsylvania State University. She did her Ph.D. in Food Technology and Microbiology at Clemson and her Masters at University of Connecticut in Pathobiology and Bacteriology. Kathy is a founding advisory board member of NMPAN and she has been a huge help to us in understanding food safety strategies and regulations in small meat plants.

Martin Bucknavage is Senior Food Safety Extension Associate also at Penn State. He has 15 years of technical management experience in the food industry from quality systems to operations to regulations. He teaches HACCP food safety, food defense and more. He has a MBA (Masters of Business Administration) from Penn State and a Masters of Food Science and Technology from Virginia Tech. We have a lot of expertise here.

The presentation is in four sections. Cathy is going to do the first two and Martin will do the second two and we'll take questions after each section. Cathy, take it away.

Presentation by Cathy Cutter

We're going to break this up into a couple different sections for Micro 101. We're going to talk a little bit about microorganisms in food and some of the specific pathogens that you guys may be familiar with. Then Martin will switch gears and talk a little bit about spoilage organisms and how one goes about doing microbial analysis.

Keep in mind that these topics are condensed versions. We spend whole semesters teaching food microbiology to our undergraduate students. We spend three days doing food microbiology short courses, and three days of sanitation short courses. This is a very condensed version.

Microorganisms in Food

To get started, we are going to focus on microorganisms in food. Specifically what are the different microorganisms that we're contending with? What are the different types? How do we differentiate them microbiologically in a lab? How does that guide us to detection and enumeration? We will look at the different types of microorganisms in food and some of the factors that affect microbial growth.

What are microorganisms? If you look specifically at the word: “micro” means too small to be seen with the naked eye, so you need a microscope. “Organism” meaning a living being. These are organisms that we can't see. Sometimes we do see them when they get to very, very high numbers in the cases of biofilms or molds or slime. But to look at these microorganisms individually, you need very sophisticated microbiological techniques such as a microscope or an electron microscope to be able to see them, such as the pictures that are listed here.

The types of microorganisms that we focus on include bacteria, which is the largest group of microorganisms and the ones that cause probably the most issues with food-borne illness. We also have molds and yeast, which are under the category of fungi. These are a little bit bigger; you can see some of these a little bit more readily. We'll talk more about molds in a couple of minutes. Viruses are very small; in fact there are viruses that are so small that they actually infect bacteria. In the scheme of things, viruses are the smallest of the microorganisms that we'll be focusing on today, followed by bacteria, and then next up is protozoa and parasites, and then fungi, molds, and yeast which can be small but they can grow with mycelium to very visible microorganisms that we'll talk about.

Bacteria are are the largest group of microorganisms. They are considered prokaryotic cells; meaning they have an outer membrane and then they also have DNA in the center of the cell. They are the most important to the food processor because they create the most issues from a microbiological standpoint with regards to pathogens as well as spoilage. Also they're the ones that we use to help with fermentation and making other foods that are fermented. Most bacteria range in size from about 0.5 microns to 2 microns in diameter. To give you an idea of how big this is, you'd have to put 25,000 cells side-by-side to equal about one inch. These are very, very small microorganisms. Again, lined up you would need quite a bit to be able to visualize.

There are different types of cell shapes, which help us in the laboratory to differentiate the organisms. They to help clue us in to which organisms we're working with. We have a broad shape, which are typically what we call bacillus in nature. We have cocci or coccus, which are spherical little round balls. Vibrio tend to be sort of spiral in nature. We also have ones that are spirillium and spirochetes, which are also types of shapes, but tend to be a little different than what we see with the vibrio or the curve-shaped rods.

Bacterial spores or endospores are also something to talk about because we'll be talking about some of these microorganisms in a few minutes. These endospores are sort of a survival mechanism for some of the bacteria that we deal with. These bacteria, when they're under these adverse conditions, like very dry environments or depending on the chemical which it's being exposed to, can revert to these spores. In the diagram at the bottom of the slide, you have what we call an actively growing or vegetative cell. When the cell is stressed, it will start to produce this endospore. What the cell does is to help direct some of the DNA into that particular endospore. The cell will then wall it off and produces this spore; then the vegetative part of it then dies off and you're left with this very resilient spore. It has protective cellular components around it.

This will stay in the spore state for a while, until there's food, moisture, the right temperature, and time as well. The cell will then go into binary fission and be able to grow and divide into actively growing a vegetative cell, which gets us back to the original state of this microorganism. Again, this is all under control of the cell given the type of environment, the food, and everything that's available. Most of the bacterial genre that are spore formers are bacillus and clostridium and you've probably heard about these organisms. As I mentioned, they tend to be resistant to heat, drying, as well as some chemicals. And again, once the conditions are right, they'll start to grow into those vegetative cells.

We also can differentiate bacteria by the makeup of the cell wall. There was a scientist back in the 1700-1800s named Christian Gram who developed the Gram Stain technique using different staining procedures. This is a way that we can differentiate between Gram-positive bacteria vs. Gram-negative bacteria. As we talk about the different organisms a little bit later on, you'll hear this terminology again.

The rationale this distinction is that the Gram-pdositive bacteria pick up certain stains, such as crystal violet, because they have a thick layer of peptidoglycan, a polysaccharide. It picks up the stain very nicely and when it does then the cells can stain a very dark purple or a dark blue.

Gram-negative bacteria, on the other hand, are a little different in their outer-membrane makeup. It's a little bit more complex; there are a lot of lipopolysaccharides and other proteins there. But it has a very thin layer of peptidoglycan, so it doesn't pick up the crystal violet near as well. Because of that, these Gram-negative cells tend to not pick up the crystal violet, but we counterstain with something called safranin, which actually gives the cells a pink color.

Why is this important? In the next slide, this schematic demonstrates the fact that we have this very thick outer membrane in the Gram-positive cell of peptidoglycan, and then a very, very thin layer in the Gram-negative cell. Because of that, the Gram-positive cell picks up the crystal violet better. But this thick layer also gives the Gram-positive organisms resilience, which makes them a little bit more difficult when it comes to treating them with antimicrobials or trying to heat-treat them. In some cases, we find that there are compound treatments that work really well against the Gram-negative bacteria primarily because chemicals and things can get in and heat can penetrate the Gram-negative cell a little bit better than they can in this Gram-positive type cell. This is also important from the standpoint of sanitation, if you're looking for chemicals or antimicrobials, you want ones that are very specific to Gram-positive organisms and those that are specific to Gram-negative bacteria.

Again, this is the terminology that we use to differentiate the bacteria. There are other stains and things that you can use for molds and yeasts and so on. But for the purposes of this exercise and this discussion, the Gram-positive and Gram-negative are what we use to differentiate bacteria.

In the case of the micro-lab, for example, if you get a Gram-positive organism, you can look at that under the stain and match that with the morphology to determine whether it's cocci or spherical in nature or if it's a rod; and that helps us as microbiologists differentiate and determine what organisms we're working with. We can then make the right decisions about compounds, treatments, and so on.

As I mentioned, the difference is between the Gram-positive with the crystal violet so you see the color differential here with the dark purple rods [see slides]. The Gram-negative tend to stay red with the safranin that gives us these pink or red colonies [see slide]. This is how we differentiate under the microscope.

As I mentioned earlier, bacteria divide by something called binary fission. The DNA is in the cell, so during binary fission the cell will turn on the components that allow for the duplication of the chromosome when the cell is ready to divide under the ideal conditions. You get the duplication of the chromosome and then the cell will then continue to grow and start to divide into two identical cells. This is what we call the binary fission. The microorganism (in this case a bacteria that grows and divides) becomes two separate cells. These two separate cells can then grow and divide again to become four. Then four become eight; and then eight become sixteen and so on.

One thing to keep in mind is that division occurs when there is proper food, proper time, proper temperature and the cells are happy. If they have to expend a lot of energy to grow, it can sometimes be to the detriment of the cell, or if there's not enough food and moisture the growth can be slowed considerably. But it's also important to know that under ideal conditions, some of these bacteria can grow from two to four to eight in as few as twenty minutes. That's why it's important to understand how bacteria grow and why it's important that we control their growth so they don't grow rapidly. It's also important to realize that if you have a high number of cells to begin with, those 1,000 cells will become 2,000 in twenty minutes. It's really important when we talk about controlling bacteria growth that we try to limit the amount of growth that these cells undergo. Reducing temperature, taking water away, doing some of the processes that we do, adding antimicrobials actually slows down this growth considerably to keep the bacteria from actively growing and getting to high numbers that cause spoilage issues or food-borne illness.

This bacterial growth curve demonstrates very nicely [see slide]. If and when the bacteria come in contact with their environment, they undergo what we call "lag phase"; where cells are sort of adapting to the environment, they're sort of sensing the amount of moisture that's available, the temperature, the food availability. Then if the conditions are right, then the cells will turn on and start to grow through binary fission.

When we're working in food processing, and in the case of spoilage and pathogens, we want to control microbial growth and keep it in this lag phase as long as possible. We know that bacteria are there in the food system, so this lag phase is really, really important to keeping the bacteria slow growing, keeping their metabolism very low. Once they start to take off and start to grow, those two cells become four, become eight and so on and this is the logarithmic or exponential phase. When this happens, we see a lot of issues with bacterial growth and we lose some ability to control the microorganisms. This actively growing phase is where the cells are using up the nutrients in their environment. They are also excreting metabolites as the result of their growth. If you have cells that are growing and excreting their metabolites, you get to a point where they've expended all of the energy in their environment. They reach what we call a stationary phase. This is sort of like a plateau in bacterial growth.

The reality of this phase is that the cells are growing and dying at the same time because of the metabolites that are accumulating in their environment. When you have death, or more death than you have growth, then you get what we call the death or logarithmic decline phase. That is where the cells start to die off as the nutrients are depleted and they can't grow and/or metabolize because it may inhibit their growth. Sometimes these metabolites are acids or other compounds that are detrimental to the bacteria in their environment.

When it comes to food processing and controlling spoilage organisms, we want to do everything in our power to control the growth in this lag or exponential phase; we don't want them in stationary phase because these are the really high levels. We want to do everything we can to control them in the lag phase.

I hope this is a brief overview of bacterial growth and binary fission and what can happen when things go wrong. But keep in mind when we're talking about fermenting organisms, that we want organisms to grow to a high enough level to be able to utilize the nutrients (i.e., glucose) to produce the lowered pH. We do want some of these starter cultures to grow very quickly to the exponential phase to get that pH drop, especially in your fermented sausages and your fermented foods. You do this by adding dextrose to your formulation, giving them sufficient nutrients so you can get a high enough level to get the pH drop the achieve those desired characteristics that we see with fermented products.

That's a quick overview of bacterial growth. We'll switch gears now and talk about mold. As I mentioned, these are a little bit larger organisms; they're multi-cellular. They have microscopic filaments called hyphae which give them their elongated structure which that you can see. Sometimes you see these on products from an undesirable standpoint when it comes to spoilage. We also see them in some fermented meat products that actually impart some flavor and desirable characteristics. We can see the mass of intermeshed hyphae microscopically; the ones that we can see visually on our food products are the mycelium.

Whereas bacteria propagate by binary fission, molds propagate by producing spores. You can see that in the top picture [see slide]. These spores are the primary agents for the dispersal of mold. You can add desirable ones to your food product or they can contaminate your food product. They can be transmitted by air, insects or animals, even rodents.

What are some of the concerns of molds in foods? Obviously spoilage. We know that there are some fermented meat products that support the growth of molds because of their low water activity, their lowered pH. Because of this, they can support these certain molds. They might come from the environment, they might come in from other ingredients that you have in your plant. The concern here is primarily the form of mycotoxins, such as aflatoxin and patulin, which these molds can produce in certain foods. They also cause allergies and upper respiratory infections. We have to be careful of the molds that are desirable, but also those that are causing spoilage and other issues that have some potential health effects.

Yeasts are in the same group of fungi as molds. But they're non-filamentous fungi and tend to be unicellular. They tend to have small round, spherical structures. You can see lots of them here [see slide]. They reproduce mainly by budding so the cells will create a bleb on the end of cell and then sort of pinch off and produce this other yeast or unicellular organism. With yeast we tend to see more issues of spoilage than anything else. We also see them in frankfurters and ham, especially in dry meats and other low-water products such as honey and jam, jellies, syrups, candies even, other fermented products as well where the water activity and pH have been lowered.

We have seen this as an issue. From a microbiological standpoint, it's really important that when you have an issue with spoilage and you're trying to figure out which organism is involved, that you have somebody that can work in the lab who can identify those microorganisms; differentiate whether you're dealing with bacteria, fungi, or what have you.

As I mentioned earlier, viruses are the smallest entity that we will be talking about. These are sub-microscopic; there are viruses that attack bacteria. These are the smallest of the microorganisms that we contend with. They're parasitic in that they need other living cells on which to grow and multiply. They're acellular microorganisms which are primarily composed of nucleic acids, which is DNA or RNA inside a protein coat.

There are a couple different types of viruses out there. This just gives you a schematic of one where the DNA or RNA is in the head [see slide]. It's got a collar and a sheath; it's got tail fibers that allow it to attach to either the mammalian cell or the bacterial cell to be able to inject the DNA. That's how some of these cells work; they get on the surface and then use the collar sheath to push the DNA into the bacterial or mammalian cell surface. Then they take over the DNA of the cell and start to replicate.

We talk about viruses because we have to be cognizant of the fact that we have employees that can be carriers of these enteric viruses such as Hepatitis A, Norwalk and other related viruses and rotaviruses. We do know that these viruses can be transferred via food that's why it's always important to have good personal hygiene, hand washing, those kinds of things because they tend to be transmitted to people via the fecal-oral route. I should mention here the fecal-oral route is when individuals who don't wash their hands very well can transmit the viruses or the virus particles through fecal material or upper respiratory secretions via their hands, by touching other inanimate objects and then somebody else touches them later on. As I mentioned, you can also transmit these viruses via food as well. Good personal hygiene, making sure people wash their hands, anybody who is sick or infected with viruses should not be working with foods, and working with employees to make sure that doesn't happen.

I want to switch gears a little bit and talk about some of the factors that affect microbial growth. These can be broken down into what we call intrinsic factors versus extrinsic factors. We'll talk a little bit about each one of these very briefly. This is primarily what we see with yeast, molds, as well as bacterial organisms. Let's spend a little bit of time on each one of these.

We know that foods themselves are excellent sources for humans and mammalians food sources: energy sources, nitrogen sources, and minerals. This is exactly what the bacteria, fungi, and yeast eat as well. They've got a sufficient amount of carbohydrates, amino acids, you name it. Foods, especially meat and poultry, are excellent sources for these microorganisms.

The other thing we need to keep in mind from the standpoint of factors that affect microbial growth is the pH. Keep in mind that the optimal pH range for most bacteria is near-neutrality. If you look at a scale of pH, it's usually goes from 1-14 with neutral in the middle. So sometimes you have a range of about 5.5 or 6 to about 7, 7.5, or 8. Interestingly enough, this is where we see a lot of bacterial growth and these microorganisms grow in that neutral range. Some bacteria such as Listeria and some other acid-loving organisms can actually grow in pHs of about 4 or so. Yeasts tend to grow at lower pHs of about 2, but they also have a range of up to about 8. Molds on the other hand have a wide range. They can grow very, very low down to about 1 pH all the way up to 11, so a very acidic to a very alkaline state. Again, from a pH standpoint, acidity is down below 7 and alkaline would be up above 7 in the 8-11 range.

Another factor is the temperature at which these organisms grow. We can classify microorganisms based on what temperature they grow at best. Whether they're growing at refrigeration temperatures or body or room temperature, or at even higher warmth temperatures. We know for example that psychrophiles will grow at about 0 degrees C, about freezing temperature; but they can also have a range of growth of upwards to about 15 degrees C (or about 9 degrees Fahrenheit) or lower. Psychrotrophs grow a little bit lower than what we see with the psychrophiles, but they can grow at room temperatures up to warmer temperatures. These are the ones that we see cause the most issues with spoilage and a lot of our refrigerated food products.

Mesophilic organisms grow very well at or near human body temperature. These are the ones that grow at a range of about 20-45 degrees C (or 68-113 degrees Fahrenheit). Keep in mind that most of the foodborne pathogens that we're dealing with (E. coli 0157:H7, Salmonella, Staphylococcus aureus) they are the mesophilic organisms. But some of these organisms like Listeria monocytogenes can also grow in different ranges, so they can grow at the colder temperatures but also grow at room or body temperatures as well.

Last but not least, there are thermophiles, which tend to be high temperature or heat-loving microorganisms. They tend to grow at much higher temperatures from upwards of 55-65 degrees C (or 131 to 149 degrees Fahrenheit).

Another factor affecting growth is oxygen availability. Aerobic microorganisms are those that require free oxygen or the ability to use free oxygen and are able to grow. We see a lot of the aerobic microorganisms in spoilage, such as what we see with pseudomonas. This might be that [inaudible, but typically characterized as sweet or fruity] smelling organism that you see on your fresh meat that sits in the refrigerator for a very long time. These would be in packages where you have a lot of air exposure, so they might be in tray racks or packaging where it's not vacuum packaged.

Anaerobic microorganisms, on the other hand, grow well in the absence of oxygen. These are the organisms such as Clostridium perfringen and Clostridium botulinum. They do not thrive very well on oxygen, but you can get some lack of oxygen in some meat and poultry products just by virtue of the cooking process, also reduced oxygen packaging systems.

Then there are organisms which I kind of call the “in-between” ones. They can grow either with or without the presence of free oxygen. One of the best organisms that we see is Lactobacillus species. It's interesting that if you take something like chicken, and you vacuum package that whole raw chicken, initially you'll have pseudomonas on there but as the oxygen is pulled out and as the microorganisms adapt, the Lactobacillus in some cases (depending upon the food product) can grow and overtake and start to create the spoilage issues that we see with some of the vacuum packaged products. Keep in mind that just by removing the oxygen, you change the atmosphere to the point where you can change the type of bacteria that will grow. That's why in some cases like vacuum packaged ready-to-eat meat products like hot dogs and similar products, if you store them for very long periods of time in a refrigerated environment, you might get more spoilage by Lactobacillus, which would give you kind of the milky white sour smelling exudate that you might see in your package over time.

Another factor to talk about is water activity. This is technical information: the vapor pressure, water and food divided by the pressure of the pure water at the same temperature. From a simplistic standpoint, it's the amount of free water that's available for microorganisms to utilize and grow. This is important because bacteria, fungi and yeast need moisture to be able to grow and replicate. When it comes to things like fermented sausages, dried sausages, jerky, those kind of things, when you remove the water, you are lowering the water activity level, or the amount of free water that's available.

You can also control the amount of free water available by adding things like solutes, which are sugars or salt that help bind the water. In the case of fresh meat products, you might have a high water activity of about 0.95 to about 0.99. Whereas in a dried product like jerky, your water activity gets down below 0.85 or even lower depending upon the jerky product that you're working with because of the drying process, the addition of salts, and other steps in the process.

In the scheme of things, if you look at the water activity in things like dried milk which has been spray dried, the moisture has been pulled out of there and you have a very low water activity of about 0.2. It goes to a scale of 0.2-1. Water is at 1. Any very dry product, very shelf-stable, has a lower water activity, again either because of the drying process or because of the addition of some of these water-binding compounds. This gives you an example of all the different products that are out there to give you a framework with which to understand water activity and why it's important. Having water there allows for microorganisms to grow. By pulling them out, drying, or binding, you actually make the water less available for the microorganisms to grow.

What are some minimum water activity requirements for microbial growth? Bacteria tend to like a range of about 0.9 - 0.99 water activity for growth. Yeasts need a little less water activity, about 0.87. Molds are much lower, down to about 0.70 in order for these organisms to grow. Keep in mind that if you have something like a fresh beef strip and then you dehydrate it or you add teriyaki or soy sauce to that particular formulation, we tend to see spoilage maybe due to mold. This is why: because they tend to survive much better and can grow on products that have the lower water activity. 

Some of the other factors to consider are the additions of chemical preservatives or other inhibitory substances to our formulation. We do this as a way to inhibit growth of microorganisms. Things including benzoate, which we add to control mold growth in some foods, or sodium lactate or sodium diacetate to control Listeria growth in some ready-to-eat products. We also use a number of different products to kill microorganisms. Again, we want to make sure that we're picking the right chemicals to kill the right microorganisms. As I mentioned earlier, have an idea of the microorganisms that you're contending with. So in a ready-to-eat environment, you're probably dealing with Listeria monocytogenes as an organism to control, therefore you need to identify sanitizers that are very specific to controlling that pathogen. You might have different sanitizers for different parts of your plant. You might use a chlorinated compound or some other compound to control the Gram-negative organisms. Again, in this case I strongly recommend working with your chemical manufacturer to come up with the right chemical for the right microorganism in the right environment.

In addition to the different growth factors we talked about: temperature, pH, and water activity; we also have to keep in mind microbial load. The temperature of the product that we're working with and then how much time it takes to get the microbial growth to grow. We also have to keep in mind the effects of biofilms. We know that biofilms do occur in food processing environments. We know that Listeria monocytogenes is one of those organisms that has the capability, as do Salmonella, to produce a film that allows them to thrive and survive in food processing environments in the right conditions.

This next slide shows the schematic of influences of pH and water activity on the selection of and growth of different microorganisms. If we look on the y-axis on the water activity, which goes from about 1 all the way down to 0.7, and then we look at the x-axis with pH of 3 up to 7, you can see that a change in water activity and a change in pH will allow for the growth and proliferation of certain bacteria. In this case something that is upwards of a neutral pH of about 7 with a lot of water in the product is going to support a lot of the vegetative microorganisms or forming bacteria that we see. Just by virtue of dropping the pH acidicly, you can then change the microfloras to support the growth of Lactobacillus or Lactobacilli. By dropping the water activity, you are now selecting for organisms that can maybe survive in a less water environment such as Staphylococcus aureus. Then we have organisms that are very salt loving, the halophilic bacteria, which can still grow in this neutral pH range, but just by virtue of dropping the pH to 4 or 5, then we're allowing for the growth and support of molds and yeast. Again just by changing the water activity or the pH in our processes we can support different kinds of bacteria that can occur in our food product.

It looks like we have a couple of questions.

Q: Cathy, that was so much great material and I'm thinking, "How are we going to get through all the rest of it?" {Laughs} Someone named Ryan Lee here in our chat box has been handling a bunch of the questions and it's just great, Ryan, thank you so much. There is this question from Traverse City, Cathy about bactericide rinses.

Cathy: Right now we know that there are phage for control…there's been some research and phage that have been used to control Listeria that you can buy commercially. There are companies out there that are doing work with phage for controlling E. coli O157:H7 and these other pathogenic E. coli on food surfaces including meat surfaces. I don't have the companies' names right off the top of my head but they are working on this. I am not aware of any commercially available carcass rinses with phage. My understanding now is it's just the Listeria monocytogenes phage control measures.

Pathogens

The first organism we want to talk about is Salmonella. This organism is a concern for meat and poultry primarily because we've seen a number of issues associated with this pathogen. We know it causes what we call a "food borne infection," which means individuals who get food borne illness from Salmonella will have a number of different symptoms such as fever, vomiting, and diarrhea, which can lead to dehydration. Primarily because the cells are ingested, the organisms will grow and divide in the gastrointestinal tract and then cause these symptoms.

We know that the infective dose of this particular pathogen is about 20 cells to 10,000 cells. Usually the onset is 12-14 hours after ingestion, depending upon the infective dose. Symptoms can last 2-3 days. But you can get prolonged infection depending upon the dose and your immune status. Again, if you're very young or very old, or if you have some underlying immunocompromised situation such as cancer treatment or transplant recipient, your immune system is suppressed so that you wouldn't be able to combat an infection with these types of pathogens. In some cases you can get what we call “reactive arthritis” a couple months after the initial infection. We see this in some individuals who complain of achy joints and things after the initial infection. 


We know salmonella has been associated with a number of different foods including meat and poultry, undercooked eggs; we've seen it with cereal that was treated with a vitamin spray after begin processed. We've seen it in pet food as well as in peanut butter and a number of outbreaks in produce, primarily because of cross-contamination with animal waste.

Salmonella is a facultative anaerobe. It's also Gram-negative, so thinking back to the earlier slides, it likes to grow under reduced-oxygen or even aerobic conditions. But it also has that very fluid outer membrane that we saw. It can grow at about 37 degrees with a pH range of about 6.5-7, but can grow as low as refrigeration temperatures and upwards of about 54 degrees that we see with some strains. It has a broad pH range in some cases as well. We know that the intestinal tracts of animals are the primary source for this organism. We also know it can appear in products, raw meat products, egg products, contaminated produce, dried nuts and grains. We also know that insects and birds can be sources of Salmonella and some plants, depending upon how the environments handle the GMPs and those things that are implemented. This organism can also survive for long periods of times in dry foods as well as refrigerated foods. We're seeing a lot more outbreaks associated with Salmonella with a number of different foods including ground products.

There are several multi-drug resistant Salmonella strains that have demonstrated resistance to a number of different antibiotics. Interestingly enough, the overall prevalence of Salmonella with these types of strains is very low. We see the prevalence of Salmonella strains at about 4.2%, while of the prevalence of the multi-drug resistant Salmonella is much lower, about 0.6%. This is a concern primarily because individuals who are being treated for Salmonella infections will have limited antibiotics available to control the infection. This is why this organism is becoming an issue from a public health standpoint. If we have Salmonella that have resistance to antibiotics it makes it harder for the physician to treat individuals who get sick with it.

The American Meat Institute has demonstrated with some research that treatments that we use to control E. coli O157:H7 on beef trimmings as well as some of the non-O157 toxin producing E. coli or the STEC [Shiga toxin-producing E. coli] are efficient in controlling also the multi-drug resistant Salmonella. When it comes to controlling this, we know that removal of lymph nodes and hides of cattle, especially those in culled dairy cows, are important to combat the Salmonella prevalence.

Other control measures include liquid heat treatment. We've seen a number of outbreaks associated with spices and peppers and things like that we use on our ready-to-eat products and also surviving in plant environments where it's very dry.  It's really important to control cross-contamination in plants; making sure you look at your air flow, dust control, making sure there's no dust residue on equipment. Also look at incoming raw materials; maybe request a certificate of analysis to indicate that spices or anything that's going to be applied to a ready-to-eat product that's not going to require another heat treatment has been treated in some way so that it's not problematic with regard to the Salmonella coming in on that product.

Next up is E. coli O157:H7. It causes a toxin-mediated infection which means it needs to be ingested by the individual. Once inside the gastrointestinal tract, the organism will burrow into the outer wall or the intestinal wall and then create bloody diarrhea as it starts to burrow. The organism also has the capability of producing a cytotoxin, which can then transfer through the bloodstream to the kidneys and cause hemolytic-uremic syndrome, or HUS. This is a very life-threatening situation, which again, depending upon the level of organism, the infection, the immunocompromised state of the individual; this is a very deadly infection in some cases. 

The infective dose is estimated at 10 cells, usually a couple days after ingestion. Typically the symptoms last just a couple of days. Some infections lead to HUS, and then half of those individuals will require some dialyses. There is a fatality rate of about 1% for the individuals who get HUS and cannot deal with the infection and succumb to HUS. Basically in this situation, you get a back up of toxins in the bloodstream and the body can't control it; it's a very horrific disease process.

The cases here are listed here are wrong [on the slide]. I apologize. It was just a copy from the Salmonella. We do know that the organism, E. coli, is associated with undercooked beef, raw milk, as well as produce contaminated via manure, which is down at the bottom here. E. coli is a facultative anaerobe. It's a Gram-negative rod. It's also acid tolerant. They have associated some growth in some fermented products down to about 4-4.5. It doesn't have any unusual heat-resistance. Cooking at a very high temperature for a couple seconds would kill the pathogen. That's why we recommend cooking ground beef products of upwards of 165 degrees Fahrenheit to kill the microorganism. We know it's associated with the intestinal tract of cattle, as well as sheep and goats. You're probably aware of some recent outbreaks associated with venison kabobs made by some students. We do see seasonal outbreaks during warmer months as well with this organism.

But most importantly, and what's probably of most concern for most folks, is later this year USDA will classify additional strains of pathogenic E. coli called a non-O157 STEC as being adulterant in ground beef. These organisms include the O26, O103, O45, O111, O21, and O45. These are the ones in ground beef. We know that there was an outbreak of E. coli O26 two years ago here in Pennsylvania. It was the first non-O157 STEC linked to beef. As I mentioned, starting in March, we'll see USDA testing for these non-O157 STEC in ground beef. They will be using some methods called polymerase-chain-reaction to detect the pathogens and Martin will talk a little bit about that shortly.

Control for the pathogen, including the non-O157 STEC, is sufficient heat control, preventing cross-contamination, making sure that manure doesn't contaminate water supplies or come in contact during processing and slaughter procedures. Also making sure that there are intervention strategies employed on the carcasses, hides, and trim destined for ground beef production. There's a number of different methods to employ for doing these intervention strategies.

Next up is Campylobacter. This is a food-borne infection, so again it has to be ingested and create diarrhea. You can get some bloody diarrhea, cramping, also a fever. There's also Guillain-Barre syndrome, which results from this particular infection causes partial paralysis in the extremities. Infective dose is about 1,000 organisms. Symptoms can last a couple of days to a week. Between Salmonella and Campylobacter (they go back and forth) Campylobacter is considered the most common cause of sporadic bacterial gastroenteritis.

We see this with under-cooked poultry and meat, unpasteurized milk, as well as contaminated water. Many in the poultry industry are working under performance standards now for controlling Campylobacter. Some of you might be aware of those different performance standards. It is a microaerophilic (low oxygen) organism, which is different from some of the organisms we've talked about earlier. It needs really, really low amounts of oxygen to survive. It's quite difficult to grow in the laboratory. It's also kind of got a spiral shape to it much like you saw in one of those earlier slides. It's motile, so it's got flagella that allow it to move around very well in the gut.

It doesn't like to grow below 30 degrees C, but we do know it can grow very slowly. It's also very sensitive to drying which is why some of the poultry industry has gone to doing air chilling because it does do a good job of knocking down Campylobacter on poultry carcasses. Chlorinated compound heat treatments are very effective at controlling this organism. We see it a lot in livestock and wild birds as well as in water and other rodents in the farm environment. Again, just good old heat and chlorinated compounds will work very well for controlling Campylobacter.

Next up is Listeria. This is a food-borne infection. It can cause diarrhea but it also can transverse the blood-brain barrier and cause meningitis, encephalitis, blood-borne infections, septicemia, and also miscarriages and stillbirth, so it can cross the placenta as well. We know that the risk-groups associated with this organism include pregnant women, neonates, and immunocompromised individuals. We know symptoms can develop a couple of days after ingestion and can be prolonged upwards of 70 days. We typically see transmission via post-processed handling, after cooking and before packaging. Listeria can sort of sneak in so it's really important to have good sanitation in those ready-to-eat areas or some kind of post-packaging treatment to control Listeria on the product. We know that some of the sources of the outbreaks recently have been deli meats, ready-to-eat meats, hot dogs, cheese as well as ice cream and we've had some outbreaks associated with cabbage and coleslaw.

Listeria is a Gram-positive organism so it tends to be a little hardier organism. It's not a spore-former. It's facultative so it can grow under reduced-oxygen environments such as we see with the vacuum-packaging materials. It's also motile. It's associated with animals, soil, water, food plants, anything that's cold and moist. Plant environments where you have a lot of cold damp areas are ideal for Listeria to grow in. As I mentioned earlier, it likes to grow at those lower refrigeration temperatures. It also can grow at very low pHs. It can also grow at water activity levels down to about 0.93 and also can survive at water activity of about 0.83.

Keep in mind that if you're using brine solution in your chilling of ready-to-eat products, you may be using a brine solution upwards of 10-12% salt solution to help cool down your ready-to-eat products. There are studies that have demonstrated that Listeria monocytogenes can survive in these high salt solutions. They may not grow but they can survive and create issues: potentially cross-contamination in your plant.

The good news is that high temperatures, typically of upwards of 150 degrees, will kill the pathogen relatively easily, just like we do for general cooking purposes. Control measures to prevent harborage sites and points for cross-contamination, with some attention to biofilms, that slime, that you might see or feel on some of your surfaces. Listeria can survive in this and transmit to other points in the process. Also, because it comes in on boots and shoes and equipment and wheels, it's really important to evaluate your transfer points in your plant. Where are people commingling? Where are carts coming from? If they coming from a ready-to-eat area and going into a raw area, then you need to clean those wheels off before they go back to the ready-to-eat area.

When controlling and monitoring your environment for Listeria standpoint, it’s important to be sure you have proper temperature and moisture control for your foods. Getting rid of that moisture, any standing water in a plant; remove that so that the Listeria doesn't have that moisture to allow it to grow. Keep things as cold as possible to keep the organism growing slowly. One other thing about Listeria to keep in mind is that it doesn't die during freezing; it can survive. Freezing just sort of puts it in an animated state. What happens is when the ice crystals thaw and you have moisture, the bacteria can start to grow again. Keep in mind, freezing isn't going to do much to something like Listeria. If you think about that outer membrane and that peptidoglycan layer, ice crystals and things have to work really hard to penetrate and that's a pretty thick layer for freezing to do anything to for the standpoint of Listeria.

Staphylococcus aureus causes a different kind of illness; it's called an intoxication. In this case, Staph will grow on the food surface if there's major temperature abuse. This will cause vomiting, nausea and abdominal cramps in individuals. Symptoms usually take a couple hours after ingestion, but they can last upwards of two days. Typically we see outbreaks associated with foods that have been temperature abused, allowing the toxin to survive. Even if you cook that particular product, the toxin remains stable; the bacteria will die, it will not grow, but the toxin remains stable and the ingestion of that toxin then causes the illness that we see: the vomiting and nausea.

We've seen a number of issues with batter or ready-to-eat products: cooked chicken, canned mushrooms, and also pastries. It's a cocci, as you can tell from the picture. It grows under aerobic conditions and it can even grow under some anaerobic conditions. It has an ideal toxin growth between 40 and 45 degrees C. As I mentioned, it is heat stable. It's also very salt tolerant, so it can grow at low water activities and survive on some food products. The primary reservoir for this organism is skin, feathers, you name it, and hides. Control methods include good personal hygiene, washing hands, if you have wounds, making sure they're covered, keeping proper control of foods and keeping hot foods hot and cold foods cold.

Onto our spore-forming organisms: Clostridium perfringens causes a toxin-mediated infection. Typically we have to have very high levels of microorganisms to cause disease but we see this primarily with temperature-abused foods such as roast beef, stews, meat, gravy, and poultry. It also is Gram-positive, spore-forming, and likes to grow under anaerobic conditions. Spores can survive boiling, so that's why it's very important after we cook something that we cool it down as quickly as possible to keep those endospores from germinating into vegetative cells, growing and producing a high level of cells that can create the disease. That's what stabilization is all about and why we have to control these organisms to the best of our ability.

There are a number of other bacterial pathogens that I won't have time to go into. They're definitely ones that might be of interest, depending upon the products that you're working with.

Viruses: we talked a little bit about them. Hepatitis A we see spread through the fecal-oral route. We see it associated with ready-to-eat foods, especially by employees who don't wash their hands between going to the bathroom and then handling food. Good personal hygiene is critical here. Norwalk virus: very low infective dose. We see a lot of the vomiting, diarrhea, dehydration that we see with virus infections. Again, we see this with some ready-to-eat foods, we see it with shellfish in contaminated water. Washing hands and wearing gloves properly is important.

Parasites also are of concern. We need to talk about this because we're dealing with animals. Giardia,Cryptosporidium parvum, and Cyclospora. We tend to see these in foods that have been contaminated with animal manure or water that's been contaminated with manure and individuals who have infections. Basically control of this is to use potable water and making sure that we have good personal hygiene, and anybody with an infection is not working with food.

That is Pathogens 101 really quickly. I see tons of questions.

Q: I think actually most of them have been covered by other great folks on the call who were able to answer them.

Cathy: Thank you, guys.

Presentation by Martin Bucknavage

Spoilage Organisms

We'll start talking about spoilage organisms. Of course a lot of what we focus on in meat is pathogens: E. coli and Salmonella. Spoilage is also important and we've spent a lot of time here and in my earlier work at ADC with {inaudible} who was on here earlier (or still on here) dealing with spoilage organisms; a very important part of the meat industry. There's a number of different type of spoilage organisms; there's groups of spoilage organisms. We're just going to briefly mention some of these and point them out. We've got a lot of questions over time and run into problems with them.

One of the first are lactic acid spoilage bacteria. There's a number of different species that can cause spoilage by bacteria. I think the important thing here is the fact that when you're doing regular testing, a lot of people are doing APC testing, they're doing coliform testing, and they see the product is starting to spoil early. A lot of times, it's lactic acid bacteria. I think the important thing is the fact that people don't really look for lactic acid bacteria. Using an APC count really is not going to allow for someone to see this type of spoilage. There's a couple of things that you have to do, such as use a more fastidious media, or a richer media for these fastidious organisms, like MRS. The other thing we have to do is to incubate these at a lower temperature. Instead of having the laboratory incubate plates at 30 or 35 degrees, we want to have these plates incubated at a lower temperature. Again, if you're seeing spoilage and your APC counts are not picking it up, one of the organisms to consider are lactic acid bacteria and in order to see them you have to change your testing methodology.

Another spoilage organism is Gram-negative psychrotrophic organisms; we kind of talked about this a few minutes ago. You'll start to see these things pop up as off flavors, especially when you start to see fruit-type flavors, or even off odors in the product. Again, if you're starting to see this type of spoilage, you have to go back and look in your operations and see where within your operation you might have some thicker issues.

Another organism that we've had a lot of issues with recently has been psychrophilic clostridium. A lot of times, processors who are vacuum-packaging a product, especially a product that has very low or no preservatives such as natural products, are starting to see more of these psychrophilic clostridium. These organisms are in the meat, there's nothing there to prevent their growth, and we start to see spoilage such as when the packing starts to blow up, the product starts to darken up. If you're doing an APC counts, you are not going to see this. In fact, finding these organisms is very difficult. Some specialized labs can go out there and figure out what this spoilage organism is. We've done a lot of work here, I know Steve Goodfellow has also done a lot of work in terms of looking for this particular organism.

We already covered yeast and mold spoilage. Again, we see these, especially yeast, in cleaners and packaged products, fresh products, and refrigerated products. We see a lot of mold on jerky-based products.

I think one of the important things that we see in plants is when we start to see mold growth on some of the ingredients that they've purchased. A lot of times the question comes up, "Hey, can I still use this?" One of the big issues when it comes to mold is the fact that a lot of molds produce mycotoxins. These mycotoxins are dangerous to people when they consume them; they can be carcinogenic and mutagenic. Remember that a lot of times these mycotoxins are heat stable. If you see some of your ingredients showing mold, the best thing to do is go ahead and throw them out.

Microbial Analysis

We're going to next talk about microbial analysis. I think there's been a lot of emphasis placed on microbial analysis to understand what your laboratories are doing. We send out samples to different laboratories and it's important for the laboratories to be doing an analysis that's an approved analysis. I know that we here at Penn State, as well as a lot of the other extension universities throughout the states, can help you if you have issues looking at the type of analysis your lab is doing to ensure that that analysis is going to be approved by the USDA. It's important when you're looking at these things to contact your laboratory, get a copy of the analysis that they're doing, and check it out to make sure that the analysis that you're doing is something that the USDA is going to accept.

One of the things that we were asked to talk about in this presentation is the CFU, such as different types of counts, whether it's APC counts or coliform counts. When we talk about CFU, we're talking about colony-forming units. To start off there is an initial dilution with the samples taken. It’s ground up in a particular diluent, and from there the sample is diluted into different test tubes or diluted out. Each time it dilutes out, it creates a factor (in this particular case, a factor of 10). Then a sample, either 1 mL or 0.1 mL, is dropped onto a plate and then those plates are incubated. Then they count the colonies to come up with the colony-forming unit.

We see a lot of times in microbial specifications if they require a 50,000 per gram CFU, what they're looking at is diluting the sample out, weighing it out on the plates, and then counting those colonies to figure out what the CFUs of those are. We also see this with E. coli. People will do E. coli counts, generic E. coli counts, in the same way. Using one type of media (they use a different media), and count the colonies on those plates to figure out the CFU per the original sample.
When we talk about APC or SPC, this is a generalized count, oftentimes using Standard Methods Agar, which is incubated at 35 degrees for 48 hours. One of the advantages of the SPC or APC method is the fact that it's very standardized. If one person is buying or selling to another person, they can do an APC count and it provides a standardized methodology. However, one of the things that's important to recognize when you're doing APC or SPC is the fact that you're not going to get all the bacteria that are there. You're not going to see anaerobic bacteria, you're not going to see lactic acid bacteria oftentimes, and because the plates are incubated at a higher temperature, you're not going to see some of these psychrophilic organisms.

Some things that you may want to do: consider using more nutritious media when you're looking for a spoilage condition. You can incubate your plates anaerobically to capture anaerobes; you can also use different types of media; incubating at different temperatures. Again, SPCs are great when we're looking at providing that standard methodology, but when it comes to a spoilage organism, or spoilage conditions, a lot of times we have to go outside of the box and do some different types of testing to figure out what's going on there.

Another question to address concerns indicator organisms. Just to briefly point out, we often look for coliform counts or E. coli counts. The first thing I'll say is that there's a number of different methodologies to look at to determine coliform counts or E. coli counts; again it's important to recognize what your laboratory is doing. Often we're using 3M Petrifilm as a way to do analysis for coliform or E. coli and that's great. Unfortunately, we've had some issues with people using different media like VRB [Violet Red Bile Agar]. VRB is primarily used in the dairy industry and when you start to plate up some of your raw meat samples using VRB, it can give you some erroneously high results. It's just something to be aware of.

The reason that we're looking at E. coli and coliform is it gives us an idea of the sanitary quality of that product. If you look at this chart here [see slides], we have Enterobacteriaceae, coliforms, and basically E. coli sits within this realm. E. Coli has the strongest correlation to being of fecal origin. As you move away from it, there's less of a correlation. As an example, we're looking at different types of products; and if you have a cooked product, theoretically, you should not have any coliforms, those should be destroyed in the cooking process. Sometimes there are particular products where coliforms are naturally present such as on certain kinds of vegetables. So it's important when you look at coliform counts to understand what you're looking for, and whether your product or some of the ingredients that you're using naturally carries those types of organisms.

The advantages of looking at coliforms or E. coli are the fact that it's a standardized methodology, produces comparative results, generally it's pretty easy to perform, and it gives us a pretty good indication of sanitary conditions. However, some of the coliform counts may be not applicable to all products. There may be some products that naturally contain coliforms. It doesn't give us an idea of how clean the process was that handled those products.

Coliforms are also not a good assessment for determining shelf life or spoilage. If your laboratory is doing shelf life analysis or spoilage analysis, the coliforms in there, especially E. coli, are probably not the best estimate for that. Why do we use indicators? Again, it assesses the food safety and sanitary conditions within the facility. It can correlate to the presence of the pathogenic organisms; although pathogenic organisms may be there even though coliform and E. coli are not.

Those were some of the different plate-count methodologies: coliforms, E. coli, and APC. We're looking at specific counts of organisms. Now we're going to talk about pathogenic analysis.

Some pathogens can be analyzed through plate count methodology, like staph or clostridium perfringens. Organisms like staph, Salmonella, and pathogenic E. coli are often done through absence or presence methodologies. Here what we're doing is we're looking at a large sample size, and we collect a large sample and we go through to see whether or not the organism is there. One of the things we'll talk about briefly is N60 sampling methodologies. Besides looking at the finished product, we also look at the environment. We do environmental sampling where we go out and we look for Salmonella, E. coli or especially Listeria.

Let's talk a little bit about sampling. It's important when we set up a sampling technique or a sampling protocol that we do these things. We determine how we're going to trim the lot size of the product. Another thing that's important is to determine the location of the samples we're going to pull, the number of samples, the size of the samples, and how we're going to go ahead and composite those samples. We'll look at an example of E. coli a little bit later to talk a little bit more about sampling.

One of the important things is to define that lot. How big is the lot? The lot is the quantity of food or the food units produced and handled under uniform conditions. It should be composed of the food produced with as little variation as possible. In determining product for a lot, it normally goes from clean-up to clean-up. However, if we're looking at something like ground beef coming in to your facility, clean-up to clean-up a lot of times won't account for that lot because you may not use it all in one day and so you may have carry-over. A clean-up to clean-up lot may not work on the ingredient side as it does on the finished product side.

Another thing that's important when we're doing sampling is to have a representative sample. We draw out from a number of different spots throughout that lot. It's important to avoid bias of drawing too much in one spot or in a spot that may be influenced by the process itself. Another thing that's important is to be able to trace back all of the product within that sample lot as well.

The number of samples is another one of the things that we talk about. The larger number of samples you take, the greater the probability you have of finding that particular pathogen. That's one of the reasons the USDA has adopted the N60 methodology: to get a larger number of samples so that we could find a smaller percent-positive sample or percent-positive lot. As the units increase, the probability of accepting a defective lot decreases. This is not to say, however, that just because you do a N60 sampling, if you do have a very low positive rate of material, there is a percentage chance you're not going to find that through that sampling. As an example of that, for a 5% effective rate, the probability of accepting a defective lot is 0.05. If that number drops to 1%, the probability of finding that is even worse. Compare that to N15 samples, where the probability is 0.46 that you would miss a 5% probability. So that's about 50% of the time that you're going to miss something that's contaminated at the 5% level. This shows you the importance of collecting a larger number of samples.

Pooling samples is the combining of stomached samples or pre-enriched samples to decrease the number of tests that are done. It's difficult to determine which sample was positive when you do this type of sampling; when you pool from different lots, we especially see this from environmentally sampling. A lot of times people want to pool their plant and pool their environmental samples. The difficulty is to find out where the positive came from when you have a positive. When you composite samples, there's also the risk of diluting out, especially in a low percentage positive sample. When you're pulling samples, it's important to always validate the protocol.

Once we've determined what our sampling is going to be and we've collected the samples, the next thing we'll talk about is testing. We can look at traditional methodology using plate and biochemical tests. One of the problems is the time that's required for those tests. There's some immunological base tests that work well and we've used for many years now. More recently we've had improvement in more DNA-based methodology. We'll talk briefly about each one of these.

When we start with pathogens, we're generally going to start off with a pre-enrichment/enrichment for this product. What we're trying to do is to take a sample, and generally when we're looking at pathogens it's a very, very low defective rate in that product. We need to take that small number and increase it. We do that through an enrichment. We take that product and put it into a broth or a media that's going to allow one or two organisms to grow up to a higher number. That media is also going to limit some of the growth other bacteria. The enrichment also helps to resuscitate organisms that may have been stressed or injured. It provides some time for organisms, especially something like Salmonella that's been put under adverse conditions, to repair itself and to start to grow.
Once we've done that enrichment, then we go into our detection method. Again, we can use a cultural method, we can use an enzyme, we can use an immunological method, or we can look at a DNA-based method to do that.

One of the common methods that we use is the immunological-based method, and the one that we use most often is the ELISA-based method. We basically have antibodies coded into a cell that are specific for a particular pathogen that we're looking at. The substrate that potentially contains the pathogen is put on there and if it attaches we sandwich it with another antibody. That antibody has a little indicator on it and so if the pathogen is there, it gives us a positive result. We see this as an immunological-based system. Again, as time has gone on the specificity of these has improved.

The other method that we hear a lot about is PCR (polymerase chain reaction). These methodologies have improved dramatically and really are very specific for looking at particular types of genes that are within a cell. Here we have a sample that we put into a broth. A specific reaction opens up the cells and the markers look for specific DNA markers within the bacteria that are there. If those specific markers are there, the DNA of those will replicate those specific genes that we're looking for and then we can detect those specific genes. A example of this is when we look at E. coli looking for the spx 1 and 2 genes that are important for the pathogenicity of pathogenic bacteria. This is a great way to look for it and we see this is being used a lot especially when we're looking at pathogenic E. coli genes.

PCR is looking for specific genes in the bacteria that are there. Another thing that's common to use is Pulsed-Field Gel Electrophoresis. We see this being used by the CDC when we have outbreak strains. They get the culture that's caused the disease, they grow it out, they split the cells, and then they get the DNA from those cells and basically chop it up with some enzymes, creating a fingerprint for that bacteria. The cells are split. They slice the cells. They put it into a pattern and the pattern will look something like this [see slide]. This will be a PFGE pattern and then they can compare the different patterns to determine which bacteria are the same. That's PFGE analysis.

I just want to mention that there are different types of errors that we see. We can have false-positives; or we can have positives that we don't detect. This is always an issue when we start to talk about testing methodologies.

Let's look at a couple specific examples. One analysis for E. coli in meat. This can be done on raw ground beef, or trim that we're going to make into ground meat. Generally what we see is that the organisms are going to be at very low-levels. The issue can be in your own slaughter operation or it can be in your incoming meat to start off with. Of course, FSIS has a number of protocols for whatever you're doing; if you're looking at trim or if you're looking at ground product. Say if we're looking at trim, one of the things we're going to do is to follow this N60 format. We will go through and get a number of samples (60 samples) and we'll combine those up into a 325-gram final product. What we'll do is look for that E. coli within that 325-gram product. If it's ground meat, we can take 65-gram samples for the 325 gram total.

As far as a detective method, a sample comes out for FSIS testing as [from slide]: a presumptive positive for E. coli is that reacting to the O157 somatic antiserum. This test and reaction indicates a “strong possibility that E. coli O157:H7 is present.” The test indicates that there's a strong possibility that E. coli is present. So if your lab comes back and says that you have a presumptive positive, it doesn't mean that you have a positive. It means that there's a strong possibility that E. coli is there.

For industry testing, test results that indicate a strong possibility of E. coli is present are considered presumptive positive results. Positive results of industry testing, for E. coli using BAX [trademark name] methodology would be considered a presumptive positive result.

What are some things that you can do working with ground meat? Of course one of the things you can do is to request a certificate of analysis when a product comes in. This would mean that the supplier of that product has gone through and tested for that product and showed that it's not there. Of course, the important thing to do is to make sure that certificate of analysis was produced by a reputable laboratory. Other things you can do is use intervention methods within your facility and you can composite sample and test.

The next organism we're going to talk about is Listeria monocytogenes. We often look at post-lethality exposed products, ready to eat meat products. Often, product sampling will not detect low levels. Another problem is the fact that we want to hold and test that product until the results are clean. Again, the best thing for product sampling is to use this as a verification. Better than looking at product sampling is to focus in on environmental sampling, such as doing zone sampling in your environment, collecting samples from the environment and testing those products.

Another method that we commonly use is waste or scrap sampling. Instead of specifically looking at products. we collect waste samples that have fallen on the floor after different pieces of equipment to see if Listeria might be there.

Getting back to zone sampling: In zone one, you're generally focusing on product samples. Zone two are those areas around product samples. Zone three are areas outside of that and zone four would be areas further away that have little impact. The majority of focus of our sampling is going to be zone two and zone three. What we want to do is to push back anything out of zone two and keep everything out beyond zone three. Again, most of our focus in zone sampling for environmental sampling for Listeria is going to be out in this area.

What do we do when we're doing environmental sampling? We want to use a moistened sponge. We also want to prepare a map so we can identify each area for recording and for recordkeeping. Another thing that's important is to use a standardized testing protocol throughout. You always want to make sure we're doing it the same way throughout so that when we record our results, those results can be reproducible.

One of the suggestions I always have for after you do sampling is to sanitize those areas in the event that you do have a positive. After you've swabbed for the positive for that sample, re-sanitize in the event that that comes up positive.

One of the questions that we often get is whether we do pre-operational type sponges or whether we do operational. I think it's important to do pre-op samples to see how well the cleaning within your facility is and whether you reduce any Listeria within your environment. However, another thing that's important to do is occasionally look at operational sampling. The reason is that once equipment starts to turn and once it starts to work, a lot of times we find that Listeria can start to leak out of particular spots that we don't see in pre-operational environments. It's good to do, on time, operational sampling.

Finally we're just going to talk a little bit about verification of sanitation. When we do sanitizing, it's important to understand how well we're doing it. The best way to do this is through microbiological sampling. It establishes how well we're doing it. It provides a record for how well we're doing. We can identify trends. If counts start to pop up, we'll see that in the analysis. There are a lot of different ways to do sanitation verification. We can use swabs, we can use sponges, we can look at APC, we can look at Listeria or even Salmonella for those operations where Salmonella may be a concern.

Another thing we can do besides looking at the actual microbes is to look at ATP testing. One of the nice things about ATP testing is it gives us very, very fast results. Beyond that we can also look at food residuals. There are a number of different test kits out there that look at proteins or that look at carbohydrates that may be left on the surface after the sanitation process is finished.

What do we want to look at as far as verification sanitation? Different types of surfaces: food contact, non-contact, personnel, maintenance people that come in to your environment, QC people, people's hands, how well are they washing, you can even look at air sampling to see whether or not our air is a concern as far as spreading microbial contamination throughout the plant.

What are some keys? We establish a program. We use standardized methodology. We set up a set frequency. Identify the different areas that we want to sample. I think we go through and set up a sampling scheme, but it's important to remember that from time to time, we're going to have a random sampling of those areas, get off and try some areas that may be off our regular sampling scheme.

Once we've done that and analyzed the data for trends, any areas that pop up, we want to make sure that we follow up and re-sample those areas. There are a number of different standards for counts, especially APC on a number of different surfaces: less than 100 cfu or 50 square cm. I think any of these would probably suffice for food process environments. If you're using ATP, again, it's important to establish what your baseline is going to be through constant sampling and validating that with APC counts. I know we went through that rather quickly here, but fortunately, I finished on time.

Q&A with Cathy and Martin:

Q: With a sampling scheme such as N60 in weight, how much product should be collected? How do you collect an N60 sample when the only thing you have to collect from is one carcass or trim from one box of beef? This is an issue in trying to sample very small plants, where lots are not 1500 lb. combos of beef trimming, the trimmings from one carcass or from one box of meat.

Cathy: It's my understanding that when you have only one batch of trim you have to get your sample from that. From carcasses, does anybody else have anything? …I don't think they're sampling carcasses for N60 the way we do it for trim.

Lauren: Someone's saying here they're not sampling carcasses for N60; carcasses are usually swab tested.

Martin: Right.

Cathy: Right, swab test with a sponge for just generic E. coli and you can also use it for pathogenic E. coli as well.

Q: Does anyone know of any studies out there on intact carcass testing for 0157 rather than combo testing? Still using the N60 method, not just pulling samples from a tub?

Cathy: Well carcass sampling we don't do for N60, so I'm kind of confused.

Arion: There was another question earlier about using sample organisms as an indicator for E. coli 0157 and the person put out the question about whether it would be appropriate to use something as broad as Enterobacteria, but then other people suggested that maybe something more specific like generic E. coli might be better. Any comments on that?

Martin: Enterobacteria is too broad of a group. When you're looking for 0157:H7…I guess a lot of it depends on if you're looking at it in a post-process environment, it's different. If you're looking at it in raw product, there's no way because I think that the counts would be…you'd be reacting all the time.

Q: I've seen studies showing that swab tests on carcasses are consistently 100-1000 times less sensitive than excision testing because of the strong bonds between bacteria and the carcass protein.

Cathy: Well, I did actually publish a paper years ago that looked at excision vs. sponging and we did determine that excision was the gold standard. Rationale for using sponge sampling is that it's easier, it's less detrimental to the carcass, it doesn't devalue the carcass so that's why we went with the sponge sample. But I would agree also sponging has limitations with regard to entrapment of bacteria in the sponge matrix. We do know that there are limitations with each of these types of methods.

Q: Plant breaks down one carcass. The trimmings are used for ground beef. That is what is sampled. To get 60 samples, you use up almost all the trim from that one carcass. How can you justify taking all of the trimming to test?

Cathy: Well, it depends. I mean, what they're asking for N60 is you take the trim and you sample surfaces with a scalpel. You go in and you cut the surfaces off. You don't have to use the whole chunk. You can use just the surface samples within that end to make your N60 sampling. That's my understanding of the methodology for that.

Arion: Usually the samples are 1" wide x 3" long, x 1/8" thick.

Cathy: If you have smaller trim than that it makes it much more difficult to do. But the idea is that you can take a bunch of surface samples from a bunch of those pieces of trim, put that in to your composite sampling to send out for micro-testing. That's my understanding.

Lauren: There are other folks who are posting really useful information. A link to the NFSAS video on N60 testing. A comment here from AMP, "inspection is encouraging the processor to perform N60 sampling techniques on carcasses to verify the effectiveness on the interventions specifically related to E. coli 0157-H7…"

Cathy: But that would be incredibly time consuming if they are going to do excision samples off of a carcass. I guess I have to ask why the FSIS is requesting that kind of sampling of carcasses for N60. A carcass swab, 300 square cm, like they published in their mega-rag, while it's not incredibly ideal, it does suffice for those purposes and you can use the sponge samples to test for pathogenic E. coli.

Lauren: A few other things coming in here. The last part of that AMP comment was that generic E. coli testing is a regulatory requirement like sponge testing. You can use the results for the carcass intervention for ground beef production.

I would like to thank Cathy Cutter and Martin Bucknavage so much for preparing all of this material for us, taking everything that they do over long periods of time, and giving it to us in just a little over an hour and a half, which was just astonishing.

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This work is supported by the USDA National Institute of Food and Agriculture, New Technologies for Ag Extension project.